BIOCHEMICAL CHANGES OCCURRING DURING GROWTH OF COCCIDIOIDESIMMITIS IN A DEFINED MEDIUM

E. P. GOLDSCHMIDT, G. W. TAYLOR, AND L. J. DEMETROULAKOS

Fort Detrick, Frederick, Maryland

Received for publication February 14, 1958

The nutritional requirements of Coccidioidesimmitis strain Cash, grown in a medium con-zisting of glucose, ammonium lactate, and in--organic salts, were reported by Goldschmidt andTaylor (1958). The present paper considers someof the biochemical changes which take place inthis medium during growth. The utilization ofglucose, lactate, and ammonia was studied withrespect to growth, fragmentation of hyphae, andthe accumulation of extracellular peptides andpolysaccharide(s) during growth.

METHODS

The strain of C. immitis, growth medium,culture conditions, and the determination of pH,protein, and viable count were the same as thosepreviously described (Goldschmidt and Taylor,1958).

Culture filtrates were obtained for analyses atvarying intervals of time by pooling duplicateflasks and collecting the mycelium on a Seitzfilter. All analytical values reported were cor-rected for loss of culture volume due to evapora-tion during incubation. The filtrates were ana-lyzed for total nitrogen by standard techniques ofKjeldahl digestion, steam distillation, and titra-tion. Ammonia was determined by steam distil-lation and titration. The nonammonia nitrogenreferred to as soluble organic nitrogen was cal-culated by subtracting the ammonia nitrogenfrom the total nitrogen. Cellular nitrogen wascalculated from the difference between the initialtotal nitrogen at zero time and the residual totalnitrogen in the culture filtrates. Glucose wasdetermined by the colorimetric method of Som-ogyi (1952) after treatment of the filtrate withthe zinc sulfate-barium hydroxide reagent of Nel-son (1944). Total carbohydrate was determinedby the anthrone method of Seifter et al. (1950)using glucose as a standard. Lactic acid was de-termined by the procedure of Barker and Sum-merson (1941).

Antisera were prepared by injecting antigeninto the ear veins of rabbits. The animals were

injected 8 times over a period of 3 weeks withequivalent doses of formalin-killed cultures (ap-proximately 0.9 mg cellular protein per dose)and bled 5 days later. Precipitin titers of theculture filtrates were determined by the ringtest method using capillary tubing as describedby Boyd (1943).

RESULTS

Substrate utilization, growth, and fragmentation.The data presented in figures 1 and 2 show thechemical changes which occurred during growthof the cultures over an 8 day period. The ammonianitrogen, lactate, and glucose were completelyutilized by the 5th day. Growth, as measured byeither increase in cellular protein or cellular nitro-gen, approached maximal levels between the 3rdand 4th day. The viable count, however did notincrease until the 4th day which was subsequentto the synthesis of the cellular material. Theseresults suggest that growth occurred solely inthe mycelial phase and fragmentation occurredonly after maximum growth was reached.The photomicrographs (figure 3) show the

changes in morphology which occurred duringgrowth of the culture. It was noted that there waslittle fragmentation of the cultures prior tothe 4th day. Short chains of arthrosporeswere observed on the 4th day (figure 3b). Theaverage chain length of the arthrospores de-creased on subsequent incubation until the 6thday (figure 3c). After the 6th day, there were nosignificant changes in the microscopic appearanceof the cultures. The morphological changes inthe cultures also suggest that fragmentation wasdelayed until the 4th day when maximum growthwas obtained. The correlation of the fragmen-tation index (figure 1) with the morphologicalchanges in the cultures, as revealed by the pho-tomicrographs, was very good.

Accumulation of peptide. The presence of non-ammonia nitrogenous compounds in the filtrateswas revealed by the difference in values for totalnitrogen and ammonia nitrogen. The data of

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TIME IN DAYSFigure 1. Utilization of glucose and lactate during growth of Coccidioides immitis strain Cash. The

fragmentation index is defined as the ratio of viable count X 1O-7 per mg cellular protein.

VIABLE COUNT x 107/MLor

300 NITROGEN pM/ML VIABLE COUNT

0

200

NH3 N CELL-N aa

-a~~~~~~~~p

TIME IN DAYSFigure 2. Utilization of ammonia during growth of Coccidioides immitis strain Cash

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GROWTH OF C. IMMITIS IN DEFINED MEDIUM

nitrogen (peptide nitrogen, 5.2 mg X 0.16 =0.83 mg nitrogen; nonammonia nitrogen, 58.8,umoles X 14 = 0.823 mg nitrogen).

Preliminary experiments employing paperchromatography indicated only trace amounts offree amino acids in the filtrates prior to acidhydrolysis. After acid hydrolysis (6 N HCl for20 hr at 110 C), large amounts of many aminoacids were observed on 2 dimensional chromato-grams employing water saturated phenol andn-butanol:acetic acid:water (4:1:5 parts byvolume).

Polysaccharide. The filtrate of 7-day-old cul-tures contained 3.8 mg per ml total carbohydrateand only 0.26 mg per ml of reducing sugar, thusindicating the presence of approximately 3.5 mgper ml of nonreducing carbohydrate. The non-reducing carbohydrate was precipitated by 4volumes of ethanol and was not dialyzable,thereby indicating the polysaccharide nature ofthis carbohydrate. Hydrolysis of the polysaccha-ride for 6 hr with 1 N H2SO4 at 105 C yieldedessentially equivalent values for reducing sugarand total carbohydrate. A number of differentreducing sugars were observed on paper chroma-tograms of this hydrolyzate, three of which wereidentified tentatively as mannose, glucose, andgalactose. The identification of the fourth sugarwill be presented elsewhere.

Precipitin antigen. Antisera from rabbits im-munized with formalin-killed cells of C. immitisgave precipitin titers of 1:100 with 7-day-oldculture filtrates. There was no evidence of anyincrease in titers with cultures incubated for 2,3, or 4 weeks.

DISCUSSION

The preceding data indicate that growth of C.immitis occurs in two distinct processes, the syn-thesis of protein to a maximum level followed byfragmentation of the mycelium. The results ofRoessler et al. (1946) suggest that this delay infragmentation was typical of their cultures also.

Fragmentation occurred after most of the am-monia nitrogen and lactate was utilized. It ispossible that the concentration of ammoniumlactate in the medium might have some influenceon fragmentation since previous results (Gold-schmidt and Taylor, 1958) had suggested thatammonium lactate might be inhibitory to frag-mentation in high concentrations. Similar resultswere observed when deionized acid hydrolyzed

casein was used at various concentrations as asource of nitrogen. These results suggest thatthere may be some inhibition of fragmentation bynitrogenous compounds, however more data areneeded to confirm this suggestion.Morton and Broadbent (1955) showed that

many fungi accumulate large amounts of extra-cellular organic nitrogen. Most of this nitrogenwas accounted for as an extracellular, dialyzablepolypeptide. They found that the production ofthe extracellular peptide decreased when the traceelement concentration of the medium was raised.

Preliminary observations indicate that the pro-duction of extracellular peptide by C. immitisstrain Cash decreased when the iron concentra-tion of the medium was increased. The signifi-cance of the extracellular nitrogen material pro-duced by C. immitis and the cultural conditionsinfluencing its formation require further investi-gation.The relationship between the extracellular

polysaccharide and precipitin antigen describedin this paper requires additional investigation.Previous studies by Hirsch and D'Andrea (1927)and Hassid et al. (1943) indicated that the pre-cipitin antigen was a polysaccharide.

SUMMARY

The utilization of glucose, lactate, and ammo-nia by growing cultures of Coccidioides immitishas been determined. The data indicate thatgrowth occurred first followed by fragmentationafter maximum growth was obtained. Fragmen-tation started after most of the ammoniumlactate was utilized.

Cultures of C. immitis strain Cash produced 5g extracellular peptide and 3.5 g extracellularpolysaccharide per L during 1 week incubation inthe defined medium.

REFERENCES

BARKER, S. B. AND SUMMERSON, W. H. 1941 Thecolorimetric determination of lactic acid inbiological material. J. Biol. Chem., 138,535-554.

BOYD, W. C. 1943 Fundamentals of immunology,pp. 365-368. Interscience Publishers, Inc.,New York.

GOLDSCHMIDT, E. P. AND TAYLOR, G. W. 1958Nutritional requirements for the growth andarthrospore formation of Coccidioides immitis.J. Bacteriol., 75, 265-271.

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HASSID, W. Z., BAKER, E. E., AND MCCREADY, R.M. 1943 An immunologically active poly-saccharide produced by Coccidioides immitisRixford and Gilchrist. J. Biol. Chem., 149,303-311.

HIRSCH, E. F. AND D'ANDREA, D. 1927 Thespecific substance of Coccidioides immitis.J. Infectious Diseases, 40, 634-637.

MORTON, A. G. AND BROADBENT, D. 1955 Theformation of extracellular nitrogen com-

pounds by fungi. J. Gen. Microbiol., 12,248-258.

NELSON, N. 1944 A photometric adaptation of

the Somogyi method for the determinationof glucose. J. Biol. Chem., 153, 375-380.

ROESSLER, W. G., HERBST, E. J., MCCULLOUGH,W. G., MILLS, R. D., AND BREWER, C. R.1946 Studies with Coccidioides immitis. I.Submerged growth in liquid mediums. J.Infectious Diseases, 79, 12-22.

SEIFTER, S., DAYTON, S., NovIc, B., AND MUNT-WYLER, E. 1950 Estimation of glycogen withanthrone reagent. Arch. Biochem., 25, 191-200.

SOMOGYI, M. 1952 Notes on sugar determina-tion. J. Biol. Chem., 195, 19-23.

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